Mast cells are central effectors in the pathogenesis of allergic reactions and play critical roles in other inflammatory diseases, such as rheumatoid arthritis. The developmental relationship of this cell type with other hematopoietic lineages has remained poorly understood. The overall goal of this proposal will be to dissect the genetic networks that regulate the selection of the mast lineage. The microphthalmia-associated transcription factor (Mitf) is essential for the development of mast cells;our laboratory has recently cloned and characterized a novel isoform of Mitf in mast cells. Utilizing in vitro mast cell differentiation systems, we have found that this isoform along with other specific Mitf isoforms are coordinately upregulated with 3 other critical mast cell transcription factors-GATA-1, GATA-2 and PU.1. PU.1 deficient cells lack mast cell-specific Mitf isoforms and cannot differentiate into mast cells. In aim #1, we will determine whether specific Mitf isoforms are targets of PU.1 by examining the regulation of their expression in PU.1 restored mast cells. To study the function of Mitf isoforms, we have developed a system to retrovirally restore Mitf expression in Mitf deficient mast cells. We have found a differential capacity for Mitf isoforms to rescue Mitf-dependent function. Aim #2 will determine the distinct biological roles for individual Mitf isoforms by analyzing Mitf-rescued mast cells for changes in phenotype, gene target expression, and capacity to repopulate mast cell deficient mice. An RNAi knockdown approach will be used to investigate the requirement of specific isoforms. Computational analysis of Mitf gene target promoters will be undertaken to identify conserved cis-regulatory elements that cooperate with Mitf to determine the mast cell program. Lastly, the biological link in mast cells between the stem cell factor (SCF) signal transduction pathway and Mitf remains unresolved. We show that SCF signaling targets Mitf for phosphorylation and that expression of the SCF receptor, c-Kit, is deficient in Mitf null mast cells. Aim #3 will investigate the role of SCF-induced phosphorylation of Mitf by testing the capacity for phosphomutant Mitf to rescue mast cell development. We will also determine whether Mitf dimerization partners, Tfe3 and Tfeb, are targeted by SCF signals. In addition, we will examine the regulation of c-Kit expression by specific Mitf isoforms and determine whether c-Kit is a target gene of Mitf. This proposal will provide insights into the mechanisms of lineage-selection during mast cell development and may lead to therapeutic targets for mast cell-related diseases.